The present invention relates to a method for the determination of .alpha.-amylase activity in body fluid, which is a clinical parameter in the diagnoses of pancreatic diseases and sialadenotropic diseases, and to reagents for the determination of .alpha.-amylase activity.
Of the methods for the determination of .alpha.-amylase activity, a method using a maltooligosaccharide has been currently in wide use with the spread of autoanalyzers. .alpha.-Amylase activity has hitherto been determined by using a maltooligosaccharide having an unmodified non-reducing terminal glucose and .alpha.-glucosidase and, if necessary, .beta.-glucosidase as adjuvant enzyme(s) for .alpha.-amylase, and determining free glucose, or by optically determining a label liberated from reducing terminal glucose. In these determination methods, an .alpha.-glucosidase originated from yeast (the genus Saccharomyces) is often used.
Since this .alpha.-glucosidase also acts on maltooligosaccharides whose reducing terminal glucose is bonded to a label, but scarcely acts on maltooligosaccharides having a glucose chain length equal to or longer than that of maltotetraose (G4), it acts very slowly on maltooligosaccharides having a chain length not less than G4 and unmodified non-reducing terminal glucose prior to the action of .alpha.-amylase, with the result that the reagent blank reaction occurs satisfactorily less. The .alpha.-glucosidase as an adjuvant enzyme needs to immediately decompose the maltooligosaccharide produced by .alpha.-amylase.
This .alpha.-glucosidase originated from yeast, however, exhibits lowered activity as the glucose chain becomes longer. Concretely speaking, the activity on maltotriose (G3) is poor. Accordingly, it is required to add an excess amount of enzyme for use as an adjuvant enzyme. In addition, the reagent poses problems in terms of stability. When the enzyme is kept in contact with a substrate for a long period of time, the enzyme gradually acts on the substrate, for which reason the reagent containing the enzyme and a substrate need to be used immediately for the determination of .alpha.-amylase activity upon preparation thereof.
In recent years, a method using a maltooligosaccharide having a modified non-reducing terminal glucose as a maltooligosaccharide for a substrate has received increased attention. This method is advantageous in that the substrate is not attacked by an adjuvant enzyme before the action of .alpha.-amylase, since the non-reducing terminal glucose of the substrate has been modified. Accordingly, reagents are still usable upon long-term storage after preparation of reagents containing the enzyme and the substrate, enabling preparation of a single-vial reagent containing enzyme(s) and substrate(s) for the determination of .alpha.-amylase activity.
Nonetheless, this method also poses problems of specificity of .alpha.-glucosidase for a substrate, and for a sensitive determination to be conducted by an efficient adjuvant reaction, it is required to add a large excess of .alpha.-glucosidase. Such use of a large amount of enzyme can cause economical disadvantages, and other problems such as cloudiness of the reagent and a possible increase of reagent blank reaction due to the enzyme existing in an enzyme standard product. Further, when a maltooligosaccharide having 7 or more glucose units is used as the substrate, maltooligosaccharides having a glucose chain length equal to or longer than that of maltotetraose (G4) may possibly be produced. It is extremely difficult to decompose such maltooligosaccharides by the .alpha.-glucosidase as mentioned above. As a result, labels cannot be liberated upon .alpha.-amylase reaction, and there was a case where .alpha.-amylase activity values needed to be calculated by multiplying measurement values by stoichiometric coefficients.
In order to solve the problems as mentioned above, U.S. Pat. No. 4,794,078 teaches concurrent use of .alpha.-glucosidase and glucoamylase. That is, glucoamylase is allowed to mainly decompose maltooligosaccharides having a glucose chain length equal to or longer than that of maltotriose (G3). According to this method, use of a maltooligosaccharide having a glucose chain length equal to or longer than that of maltoheptaose (G7) as the substrate does not necessitate the aforementioned calculation by multiplying stoichiometric coefficients, since the maltooligosaccharide can be easily decomposed into glucose units, which in turn results in improvement of sensitivity of the determination system. Concomitant decrease of the total enzyme amount can lead to advantages that the method is economical, the reagent does not become cloudy, and that the reagent blank reaction can be inhibited.
While glucoamylase acts well on maltooligosaccharides having a glucose chain length equal to or longer than that of maltotriose (G3) irrespective of the chain length of glucose units, it acts poorly on maltose (G2). In addition, since glucoamylase does not act on reducing terminal glucose having a label bonded thereto, it cannot be used alone as an adjuvant enzyme for .alpha.-amylase activity determination.
An object of the present invention is to provide a method for the determination of .alpha.-amylase activity, which can solve the problems as mentioned above.
A further object of the present invention is to provide reagents for the determination of .alpha.-amylase activity.